The present invention relates to the field of chemistry and more particularly to that of plant chemistry.
More especially, it has as its aim a new process for obtaining a titrated preparation based on algae with active principles ensuring the fixing of calcium by the osseous cells.
Its specific aim is a process for obtaining powders or extracts from the alga Padina pavonica, titrated with active principles by means of a coefficient of equivalence with estradiol, which consists of dosing the extract or the powder of Padina pavonica using a method in which osteoblasts are cultivated in a medium rich in calcium ions, an analysis is made of the calcium fixed by the cells of the extracellular matrix in the presence or absence of agents-antagonistic to the fixing of calcium, and the results are expressed either in arbitrary or international units in relation to a reference standard or by reference to the known activity of fixed doses of estradiol or of any other agent favouring the fixing of calcium.
More precisely, the process according to the invention consists of preparing a suspension of a human or animal osseous cell line, by adjusting the cellular density determined by turbidimetry or by calculating from a reference value, leaving to incubate for one to three days at 37xc2x0 C. in the presence of carbon dioxide and of a complete culture medium (CCM), adding to some wells an agent inhibiting the fixing of calcium or a calciotropic hormone, and some extract to be titrated, eliminating, the surnatants from the culture, rinsing out the wells with a rinsing medium without calcium or magnesium (P.B.S. for example) in such a way as to eliminate the non-fixed calcium from the culture medium, eliminating the supernatants fractions, and adjusting the volume of the dispersion with a fixed volume of mineral acid such as hydrochloric acid, sulphuric acid, phosphoric acid or perchloric acid or with any other product capable of destroying the extracellular matrix built up by the cells being cultured. In conclusion, the mixture is adjusted and the calcium is dosed, for example according to the standard AFNOR nxc2x0 NTxe2x88x92690.005.
The dosage of the calcium constitutes an indicator of activity which is easy to determine. The fixing of the calcium by osteoblasts or cells from a cell line is dependent on the calciotropic agents activating the fixing of calcium (estradiol, vitamins D3, calcitonin) or agents deleterious to the fixing of calcium such as inhibitors of the calcic canals (verapamil, cinchonine or diltiazem), or pro-inflammatory cytokines: prostaglandins, interleukins, PAF etc.
The extracts or powders of the algae Padina pavonica have the effect of stimulating biological activity (synthesis of proteins and glycoaminoglycanes) but also act to restore physiological functions such as the fixing of calcium by osteogenic cells. For this, the fixed calcium is dosed with a fixed cell line, such as for example the cell line UMR 106 or the line G 292, in the presence or not of deleterious agents such as inhibitors of the calcic canals and pro-inflammatory agents such as IL 1. Normal osteoblasts give good results, but their availability presents a problem. The preparation to be analysed is tested in parallel on the same cell line, in the presence or not of these same deleterious agents. The quantity of fixed calcium in relation to that obtained with the calciotropic agent and with those obtained in the presence of agents inhibiting the fixing of calcium are compared.
In fact, it has been found that the active molecules of the alga Padina pavonica (MAPP) kept their property of improving the fixing of calcium by the osseous cells, even in the presence of an inhibitor of the calcic canals or an inflammatory agent such as interleukin IL-1. This indicates that the extract of Padina pavonica possesses one or more active principles whose mode of action is different from that of known substances which are not capable of improving the fixing of calcium in the presence of a calcic inhibitor.
The difficulty of the situation comes from the fact that the activity of the alga varies greatly, depending on the season, the growth and the stage of development of the plant, the depth, the luminosity and the fact that the technician or final consumer wants a product whose activity is constantly. It is necessary therefore to produce an extract in conditions such that it is possible to be free from the variations in active principles in the plant, and the problems arising such asxe2x80x94what should be the activity of the extract and how obtain a product with a titre that is always constant?
The present invention aims to solve the technical problem outlined above. The harvesting of plants, their treatment and the manufacture of stabilised extract require the dosage of the activity in the substances treated. The gathering-in of the plant should be carried out when the active principle content is at its optimum level, so it is necessary to determine the moment. The preservation of plants before their processing, the extraction of the raw material, the manufacture of the extract make it necessary to have at one""s disposal an analytical method making it possible to quantify the activity of the material.
The production of an extract usually calls for chemical dosage of the active molecule. When the activity is brought about by a family of molecules, it becomes difficult or impossible to refer to a chemical dosage. The difficulty of the problem comes from the fact that the molecules can exist in the form of isomers or tautomers, some active and some inactive. If the isomerism is supported by functional groups, it is possible to dose the functional groups and these support the totality and the exclusivity of the activity of the active principles. We can illustrate this by citing the dosage of anthraquinones with senna, alkaloids, etc. In the case of cardiotonic heterosides of digitalis, anomalies of correlation between the chemical analysis and biological activity have been observed many times, as a result of the multiplicity of the active principles with unequal levels of activity.
If the isomerism is supported essentially by structural elements, the analysis will be more delicate and will require the characteristic structural elements to be disclosed by spectrophotometric measurement, or call for characteristic reactions such as coloration of the functions that may be representative of the molecular family being researched. For example, in the case of cis-trans isomerism, the double link should be functionalized by fixing a reactional function. On this function, it will be possible to fix a chromophore group, by a bonding reaction (isothiocyanate of fluorescein, for example). It is also possible to use mass spectometry but it should be remembered that this technique is destructive, and it will not then be possible to re-verify the activity of the fraction isolated.
In the case of numerous substances, it is usual to dose the activity by biological means, for example in the case of penicillin, numerous vitamins such as vitamin A. vitamin E, antibodies, antigens, certain hormones such as insulin, FSH, GSH, cytokins such as interferon, TNF etc. This technique is applied essentially to dosages which would be impossible by chemical means. The case of vitamins A and E provide a good illustration, since they exist in the form of numerous isomers.
The problem becomes even more complex when dealing with substances without a chromophore group that is easily detectable and whose structure is unknown. In this case, the dosage of the biological activity seems to be the sole method or at least the method of choice.
Amongst the methods of dosage of activity, one finds methods which apply to substances modifying a biological activity of procaryotic or eucaryotic cells. The dosage of biological activity poses the problem of the stability of normal cells or cell lines. Furthermore, the signal analysed may vary according to the operational criteria impossible to specify in many cases. At the time of the usage of these methodologies, the authors are confronted with the variability of response of the cells. The response of the strain should therefore be calibrated in relation to a reference system. This system does not give a good response in the case in question, as it so happens that Padina is the only known plant capable of inducing a response on the fixing of calcium in the presence of a calcic inhibitor (Inh-Ca) and in the presence of an inflammatory agent such as IL-1. All the other substances tested hitherto cannot restore the fixing of calcium in the presence of a calcic inhibitor (Inh-Ca).